The formation of a rolling larval chamber as the unique structural gall of a new species of cynipid gall wasps

Insect galls, which often have complex external and internal structures, are believed to have adaptive significance for the survival of insects inside galls. A unique internal structure was discovered in the gall of a new cynipid species, Belizinella volutum Ide & Koyama, sp. nov., where the larval chamber could roll freely in the internal air space of the gall. Observations of the live galls using micro-computed tomography (micro-CT) revealed its formation process. The larval chamber becomes isolated from the internal parenchyma soon after the gall reaches the maximum diameter and is able to roll as the internal air space is expanding from the surrounding parenchyma to the outer gall wall. The enemy hypothesis could partly explain the adaptive significance of the unique structure of the gall of B. volutum.

diameter, milky white or light brown, and is not attached to the outer wall or internal parenchyma; therefore, it can roll freely and its position within the internal air space is indeterminate (Fig. 3).

Temporal changes in the internal gall structure
Temporal changes in the internal gall structure were observed within a single specimen by micro-CT (Fig. 4).The gall interiors of the four specimens at different maturity levels were also observed (Fig. 2, bottom).The gall interior was initially filled with juicy parenchyma without air spaces, and a larval chamber with a sclerotized wall was located in the center (Figs.2a, 4a).An air space then formed between the larval chamber wall and the surrounding area of the parenchyma (Fig. 2b).A number of small air spaces arose in the parenchyma, and the central air space expanded in connection with them (Figs.2c, 4b).Finally, a large air space accounted for most of the interior of the gall (Fig. 2d).As internal maturation progressed, the outer wall of the gall hardened (Supplementary Table S1).

Etymology
The new species is named after its unique internal gall structure: a free-rolling larval chamber.The Latin word "volutum" means "rolled".

Japanese name
Insect name: kashiwa-suzu-tamabachi.Gall name: kashiwa-ha-suzu-tama-fushi.Each Japanese word means as follows: "kashiwa" = Q.dentata, "suzu" = small hollow Japanese bell that contains a pellet, "tamabachi" = cynipid wasps, "ha" = leaf, "tama" = ball, and "fushi" = gall.www.nature.com/scientificreports/Mesosoma black.Legs dark brown except for black coxae and lighter basal and apical margins of femorae.Metasoma black except for lighter anterodorsal margin of metasomal tergite II, lateroventral area, and hypopygium.Head 1.3 times as broad as high in anterior view, 2.1 times as broad as long in dorsal view, clearly broader than mesosoma in dorsal view (Fig. 5a,c).Mandible bidentate.Maxillary palpus 4-segmented; labial palpus 3-segmented.Oral foramen 0.9 times as broad as height of compound eye.Ventral clypeal margin incised medially.Anterior tentorial pit distinct.Epistomal and clypeo-pleurostomal sulci distinct.Lower face faintly coriarious, with sparse long setae; low longitudinal ridge extending medially from area between antennal rims to clypeus; facial strigae absent medially, but radiating from clypeus laterally, indicating subocular impression; distance from lower edge of antennal rim to ventral margin of lower face 2.6 times as long as distance between antennal rims.Malar space 0.4 times as long as eye height.Diameter of antennal rim almost as broad as distance between inner margins of rims and distance between lateral margin of antennal rim and mesal margin of compound eye.Transfacial distance 1.2 times as broad as height of compound eye.Direction of straight part of inner margins of compound eye slightly diverging ventrally.Gena finely coriarious, with sparse long setae, broadened behind eye, slightly visible in anterior view.Vertex and frons finely coriarious, with sparse short setae; POL 1.1 times as long as OOL, 1.8 times as long as LOL; OOL 2.0 times as long as distance from posterior edge of lateral ocellus to occipital margin in dorsal view.Occiput finely coriarious, with sparse short setae; occipital carina absent.Antenna with 14 antennomeres (Fig. 5b); relative lengths of scape, pedicel, and F1-F12: 17, 7, 20, 14, 12, 12, 10, 10, 9, 9, 8, 8, 7, 15.
Metasoma smooth and polished, longer and broader than metasoma (Fig. 6a,c); metasomal tergite II extending more than half of metasoma, with patch of sparse setae antero-laterally; projecting part of hypopygial spine broad and short, 0.7 times as long as broad in ventral view, tapered to apex, with dense long subapical setae, extending far beyond apex of spine (Fig. 6b).

Molecular profiles and phylogeny
Partial sequences of cytochrome c oxidase subunit I (COI), cytochrome b (cytb), long-wavelength opsin (opsin), and D2 loop of the 28S ribosomal RNA gene (D2) were determined in B. volutum.No species with highly matching sequences were identified in the database.The new species formed a well-supported clade with B. vicina and B. gibbera with 100% posterior probability support (Fig. 7a).

Biology
The gall is induced on the vein on the underside of the leaf (Fig. 7c), reaches its maximum diameter in August, and remains on the leaf after maturation.The pupa is observed in the gall before the end of September.The adult female emerges from the gall between December and January.Although the egg-laying site has not yet been confirmed, the egg is probably laid in the latent or winter buds, considering the developmental state of the host plant during the adult emergence season.The timing of gall formation and the morphological characteristics of the adults indicate that the above females are the asexual generation.Neither the sexual generation galls nor adults are known.

Discussion
Free-rolling larval chamber formation Cynipid galls can be divided into the larval chamber and the outer gall 11 .A simple-structured gall consists only of a larval chamber (Fig. 1d).When the outer gall is present, the larval chamber is surrounded by woody sclerenchyma or spongy parenchyma (Fig. 1a,b,c,g,h) 11 .In more complex galls, the larval chamber can be separated from the outer gall by complex internal air spaces (Fig. 1a,b).In the asexual generation galls of the two previously known Belizinella species, B. vicina and B. gibbera, their larval chambers are surrounded by parenchyma without any air space 23 .However, in the mature gall of B. volutum, the larval chamber is completely separated from the outer gall, and the position of the larval chamber within the gall is indeterminate.Although such unique internal structures with a free-rolling larval chamber have been reported in a few cynipid galls 12,24,25 , little is known about their formation process.This study visualized this phenomenon using micro-CT for the first time.Cynipid galls develop in three phases: initiation, growth, and maturation 10,12 .Formation of the internal air space occurs during the maturation phase 10,12 ; therefore, the formation of the free-rolling larval chamber progresses during the maturation phase.The gall interior of B. volutum is initially filled with juicy parenchyma without air spaces, as is typical of other galls of Belizinella.However, air spaces appear from the surrounding area of the larval chamber, and eventually, a large portion of the gall interior becomes hollow.Because the larval chamber and surrounding parenchyma are completely separated in the early stages of the mature phase, air space formation may be predestined for the growth phase.Internal air spaces are also observed in some fruits and seeds (e.g., fruits of Sapindius spp. 26).Although gall formation is thought to involve the expression of a group of genes related to flower and fruit formation 27 , it remains unclear which group of genes is expressed during complex gall formation in gall wasps 28 .It is an interesting future challenge to determine the gene expression profiles in cynipid gall formation, including the free-rolling larval chamber of the gall of B. volutum.

Adaptive significance of the free-rolling larval chamber
Nutrition, microenvironment, and enemy hypotheses have generally been proposed to account for the adaptive significance of insect galls 8,12,13 .Since the complex external and internal structures of insect galls are thought to have been acquired through interactions between cynipids and their natural enemies, parasitoids and predators 13 , the enemy hypothesis could account for the evolution of the complex structures of insect galls.Gall toughness, gall wall thickness, the number of larval chambers per gall, external hairs or spines, and internal air spaces are thought to be protective structures against natural enemies 12 .However, most insect galls are not free from natural enemies, even though they have complex internal structures such as free-rolling larval chambers 13,29 .Zhang et al. 29 reported that a cynipid gall with a free-rolling larval chamber was attacked by a single species of Sycophila (Hymenoptrea: Eurytomidae).In addition, we identified a parasitoid, Torymus sp., that emerged from the galls of B. volutum.
The genus Torymus Dalman is one of the ectoprasitoids of Cynipidae and Cecidomyiidae and has a long ovipositor that penetrates the outer gall into the larval chamber and parasitizes the larvae inside the gall 30,31 .Although gall toughness and gall wall thickness are generally expected to contribute to protecting larvae inside, their protective effects might be obscured against such parasitoids.However, in the case of the mature gall of B. volutum, it is assumed that even if the tip of the ovipositor penetrates the outer gall wall and reaches the larval chamber wall, it cannot easily reach the inside of the chamber because the larval chamber is rolled within the gall when the ovipositor forces it to penetrate (Supplementary Video S1).Therefore, the Torymus sp.probably attacks B. volutum before the internal air space of the gall is completely developed.In fact, one of the two individuals of Torymus sp.emerged from the gall in July, when most of the galls of B. volutum did not reach their maximum diameter.
In addition to insect parasitoids, galling insects are under selective pressure from predators, such as birds 32 .Although further experimental studies are required, the unique structure of the gall of B. volutum might be advantageous for avoiding bird predation.Birds can easily locate the exposed chamber if the larval chamber is attached to the outer gall, whereas the larval chamber of B. volutum may roll away through a broken gall wall when birds vigorously break it.Although we did not directly observe bird predation on the galls of B. volutum, some broken and hollowed galls remained on the plants.
It is difficult to account for the adaptive significance of the free-rolling larval chamber of the gall of B. volutum by nutrition and microenvironmental hypotheses.The known habitat of B. volutum is restricted to abandoned semi-natural grasslands that had been managed by regular burning 22 .In addition, the grassland area is covered with snow for approximately three months during winter.Although the internal air spaces of the galls may mitigate rapid temperature increases 33 , it is still unclear whether the free-rolling larval chamber of the gall of B. volutum has advantages for their survival in such habitats.

Continuous observation of the internal structure of live galls using micro-CT
We applied micro-CT to observe the internal structure of insect galls with live larvae.Micro-CT has recently been widely used to analyze the internal and external insect structures [34][35][36] .The main advantage of this method is the non-destructive and three-dimensional observation of the internal structure.Live plants are capable of continuous micro-CT in a living state 34 .It has also been shown that the continuous observation of live insects using micro-CT is possible 37,38 .In the present study, we observed the maturation phase of the cynipid gall after detachment from the plant; however, it may be possible to monitor gall development continuously from the initiation and growth phases using potted live plants.Non-destructive visualization of the internal structure of insect galls by micro-CT would contribute to future studies on understanding the adaptive significance of insect galls.Thirty galls of B. volutum, which were determined to have almost reached their maximum diameter, were collected from the field on the leaves of Quercus dentata in September 24, 2021.The collected galls were stored in moistened moss in square plastic cases with mesh lids.Those cases were kept in an incubator under the following temperature settings: 1 °C at 00:00-05:00, 5 °C at 05:00-19:00, 3 °C at 19:00-24:00 in January; 5 °C at 00:00-07:00, 10 °C at 07:00-09:00, 15 °C at 09:00-17:00, 10 °C at 17:00-24:00 in February and December; 10 °C at 00:00-05:00, 15 °C at 05:00-08:00, 20 °C at 08:00-19:00, 15 °C at 19:00-24:00 in March-May, September-November; 15 °C at 00:00-05:00, 20 °C at 05:00-08:00, 25 °C at 08:00-19:00, 20 °C at 19:00-24:00 in June-August.Galls were observed every 3-5 days.When the moss dried, water was added to keep it moist.Adult insects that emerged from galls were immersed in 99.5% ethanol.

Examination of gall structures
Several additional galls were collected from the field in August 17, 2022, for micro-CT scanning.It was conducted using an inspeXio SMX-225CT FPD HR Plus (Shimadzu, Kyoto, Japan) with a tube voltage of 115 kV, tube current of 70 µA, and a slice thickness of 49 µm.Visualization and measurement of the micro-CT images were conducted using VGSTUDIO MAX ver.3.4 (Volume Graphics KK, Aichi, Japan).
The observations and measurements of galls were performed in two ways.First, changes in the internal structure within a single gall specimen were observed at one-month intervals (August 30, 2022 and September 26, 2022) using micro-CT.Two gall specimens with different initial maturities were used in this observation.Second, four gall specimens with different maturities were selected and observed non-destructively using micro-CT in August 30, 2022.Then, longitudinal sections of the four galls were observed directly by dissection and were photographed.As an indicator of gall toughness, the force required to penetrate the gall wall was measured before dissection using a digital force gauge (ZTS-500N, Imada, Aichi, Japan) with Shiga insect pin No. 3 (Shiga Konchu Fukyusha, Tokyo, Japan) attached vertically to the tip of the instrument.The maximum force at the penetration point through the outer gall wall was recorded for all four gall specimens.Measurements were conducted using a pointed needle tip and a non-pointed needle head.

Examination of adult morphology
Nineteen adults of B. volutum were yielded from the galls we collected in September 24, 2021.Adults were preserved in 99.5% (v/v) ethanol.Twelve adults of B. volutum were air-dried and mounted on the tips of triangular papers for morphological examination, and the remaining seven adults were preserved for future studies.Adult morphology was observed and measured using a stereomicroscope (S8-APO, Leica K.K., Tokyo, Japan) and a scanning electron microscope (JSM-6380LV, JEOL, Tokyo, Japan) operating at 1.5 kV.The lengths of each body part were measured using an ocular micrometer.Focus stacking was conducted for a photo of the adult habitus using CombineZP software (https:// combinezp.software.informer.com/).All images were processed into figure plates using GNU Image Manipulation Program (GIMP 2.10.20;https:// www.gimp.org/).
The terminology used for morphological characteristics followed those of Ronquist and Nordlander 39 , Melika 19 and Liljeblad et al. 20 .The terminology used for the surface sculpture followed that of Harris 40 .The following morphological abbreviations were used: POL, postocellar line (the distance between the inner edges of the two lateral ocelli in dorsal view); OOL, ocular-ocellar line (the distance from the outer edge of the lateral ocellus to the compound eye in dorsal view); LOL, lateral-ocellar line (the distance between the median and lateral ocelli in dorsal view); and F1-F12, the first to twelfth flagellomeres.
Combined datasets of the cytb and opsin regions were created, including the aforementioned species, B. vicina, B. gibbera and other global Cynipini species presented by Fang et al. 21and Nieves-Aldrey et al. 47 (Supplementary Table S2).We did not include the COI and D2 genes in our datasets because there were no available sequences for these genes in B. vicina and B. gibbera.The datasets were divided into seven partitions (the 1st, 2nd, and 3rd codon positions of cytb; the 1st, 2nd, and 3rd codon positions of opsin; and the intronic section of opsin).The best-fit model was selected for each partition, according to Nieves-Aldrey et al. 47 .Bayesian phylogenetic

Figure 2 .
Figure 2. External and internal structure of Belizinella volutum galls with different maturity levels.(a) Low maturity.(b) Mid-low maturity.(c) Mid-high maturity.(d) High maturity.The top row shows the external structure, the middle row shows a longitudinal section by dissection and the bottom row shows the internal air spaces visualized by micro-CT (light blue).

Figure 3 .
Figure 3. Movement of a larval chamber of mature Belizinella volutum gall at different angles visualized by micro-CT.The side of the gall contacting the leaf is at the bottom in the left column, to the left in the middle column and above in the right column.Each arrow indicates the larval chamber.

Figure 4 .Figure 5 .
Figure 4. Temporal changes of internal structure of Belizinella volutum gall within a single specimen observed by micro-CT.(a) Internal structure on August 30.(b) Internal structure on September 26.The upper row shows transverse sections and the lower row shows longitudinal sections.

Figure 7 .
Figure 7. Phylogenetic position of Belizinella volutum within Cynipini.(a) Bayesian phylogenetic tree of species sampled from across the Cynipini, based on combined data of partial sequences of cytb and opsin regions.Black stars at nodes indicate ≥ 95% posterior probability support; black circles indicate 70-94% posterior probability support.(b) Habitus of B. volutum (holotype) (scale bar 1 mm).(c) Galls of B. volutum on Quercus dentata (scale bar 1 cm).
Of the 30 galls we collected in September 24, 2021, two of them yielded only Torymus sp.(Hymenoptera: Torymidae) each, 19 yielded only B. volutum, and 9 yielded no adults.Each of the two Torymus sp.emerged in July and September, 2022, under laboratory conditions.No inquilines emerged.The partial sequence of the COI gene was determined for Torymus sp. and deposited in GenBank under accession number OR339872.No species with highly matching COI sequences were identified in the database.Voucher specimens were deposited in the NSMT (accession numbers: NSMT-I-Hym 77822-77823).https://doi.org/10.1038/s41598-023-43641-6www.nature.com/scientificreports/